Ethanol has complex effects on blood vessels. Ethanol can cause both vasodilation and constriction. Constriction of blood vessels by ethanol may contribute to the development of hypertension and stroke in people consuming ethanol. The mechanism of this constriction is poorly understood. Studies in non-vascular cells and preliminary data by the PI, using blood vessels, suggests that vasoconstriction by ethanol may involve stimulation of protein kinase C. Thus, the primary purpose of the experiments in this proposal is to determine whether ethanol constricts vessels by activating protein kinase C. We will examine the role of protein kinase C in vasoconstriction by ethanol by determining whether a variety of protein kinase C inhibitors antagonize and reverse vasoconstriction by ethanol in three types of preparations: 1) isolated microvessels, 2) vascular strips, and 3) exposed microvessels in anesthetized animals. We will also examine the role of protein kinase C in the vascular effects of ethanol by determining whether depletion of protein kinase C in vascular smooth muscle cells and blood vessels reduces responses to ethanol. Activation of protein kinase C by ethanol may be indirect. Stimulation of phospholipase C results in activation of protein kinase C. There is evidence that ethanol stimulates phospholipase C in non-vascular tissues, but no one has determined whether ethanol stimulates phospholipase C in vascular tissue. Therefore, we propose to perform the first studies to determine whether ethanol stimulates phospholipase C in vascular tissue. The experiments to determine whether ethanol stimulates phospholipase C in blood vessels and vascular smooth cells will utilize several methods. First, we will determine whether ethanol stimulates production of inositol polyphosphates in vascular smooth muscle cells and blood vessels by measuring inositol polyphosphate levels in these preparations after exposure to ethanol. We will determine the time course and dose-response relationship of any effect we observe. Second, we will determine whether phospholipase C inhibitors antagonize three responses to ethanol: 1) generation of inositol polyphosphates in blood vessels and vascular smooth muscle cells. 2) vasoconstriction and 3) increases in intracellular calcium concentrations. These experiments, utilizing state of the art techniques, novel pharmacological agents and preparations ranging from cells to whole animals, will provide strong evidence regarding a new hypothesis explaining the mechanism of vasoconstriction by ethanol.